Preparation method and application of active targeting SN38-fatty alcohol prodrug nano preparation

Abstract

The invention discloses a preparation method and application of an active targeting SN38-fatty alcohol prodrug nano preparation, and belongs to the technical field of pharmaceutical preparations. According to the invention, SN38-fatty alcohol prodrugs are designed and synthesized, a series of self-assembled nano preparations are prepared, and the anti-tumor effect of the nano preparations is investigated. Results show that different modification proportions of folic acid can significantly influence the stability, cytotoxicity and antitumor activity of the active targeting type self-assembly nano preparation. According to the invention, a micromolecular prodrug self-assembled nano preparation is combined with an active targeting delivery strategy for the first time, so that the tumor accumulation of the drug is enhanced, and the safety and effectiveness of treatment are improved. Meanwhile, the optimal modification proportion of folic acid is comprehensively screened out by coordinating the proportion of the invisible molecules and the targeting ligand, and the paradox between the active targeting effect and the in-vivo and in-vitro stability of the active targeting type nano preparation is effectively solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and relates to a preparation method and application of an actively targeted SN38-fatty alcohol prodrug nanoformulation, and specifically to the construction of a sensitive bond-bridged SN38-fatty alcohol prodrug and an actively targeted self-assembled nanoformulation containing the prodrug, as well as the application of the nanoformulation in the preparation of anti-tumor drugs. Background Art

[0002] In recent years, prodrug self-assembled nanoformulations have shown unique advantages in the field of drug delivery and treatment of cancer. It integrates the advantages of prodrug strategies and nanotechnology, and has the characteristics of high drug loading, good stability, and low toxic side effects. Prodrug self-assembled nanoformulations are usually composed of four modules: drug module, response module, modification module, and surface functionalization module. Among them, the surface functionalization module usually uses PEG material as a "stealth molecule" to improve the stability of the preparation, prolong blood circulation time, and slow down the clearance in the body. Its form and function are relatively simple. At the same time, prodrug self-assembled nanoformulations mostly rely on the enhanced permeation and retention effect (EPR effect) to achieve passive accumulation in tumor sites. However, due to the vascular dependence of the EPR effect, its situation is highly variable. Studies have shown that the EPR effect is extremely heterogeneous between different patients, and even in different stages of development of the same patient or the same tumor type. Nanoformulations that rely on passive targeting have not yet achieved substantial improvements in patient survival, which may indicate that tumor patients have reduced tumor susceptibility to nanoformulations or lack of EPR effect.

[0003] Active targeting strategies based on the specific recognition of targeting ligands with their cognate receptors have brought more possibilities for the precision treatment of tumors. Various targets, such as folate receptors, sodium-dependent multivitamin transporters (SMVTs), and integrins, have been widely used in nanodrug delivery systems for targeted cancer therapy. Compared with non-targeted nanoformulations, the number of ligands modified by active targeted nanoformulations profoundly affects their biodistribution and pharmacokinetic behavior. A sufficient number of targeting ligands is a necessary condition for the efficient binding of nanoformulations to targets. However, an excessive number of ligands may also have some adverse effects. For example, (1) excessive stacking of ligands may produce greater steric hindrance, which will hinder the effective binding of nanoformulations to targets. (2) As the number of ligands presented on the surface of nanoformulations increases, the presentation of "stealth molecules" (such as PEG) on the surface gradually decreases, ultimately leading to the activation of the conditioning cascade and an increased risk of clearance by the reticuloendothelial system (RES). (3) Many ligand molecules have extremely poor water solubility, such as folic acid (0.0016 mg / mL) and biotin (0.2 mg / mL), which also brings some pharmaceutical preparation problems. Therefore, how to ensure the active targeting effect of nanoformulations while improving the stability of the preparations and avoiding RES clearance is a problem that needs to be solved urgently. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an active targeted SN38 fatty alcohol prodrug nanoformulation, which combines the small molecule prodrug self-assembly nanoformulation with the active targeted delivery strategy for the first time, and introduces active targeting ligands, such as folic acid, in the surface functionalization module, so that the prodrug self-assembly nanoformulation can bind to the specific receptors on the surface of the target cell, further enhancing the accumulation of drugs at the tumor site, and improving the safety and effectiveness of the treatment. At the same time, by coordinating the modification ratio of the "stealth molecule" and the targeting ligand, the optimal modification ratio of folic acid is comprehensively screened to achieve a balance between the stability and effectiveness of the nanoformulation, which provides a new direction and new ideas for the construction of the prodrug self-assembly nanodelivery system based on the active targeting strategy, and is expected to solve the paradox between the active targeting effect and the stability in vivo and in vitro.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a SN38-fatty alcohol prodrug, the structure of which is shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a method for synthesizing the above-mentioned SN38-fatty alcohol prodrug, and the synthetic route is as follows:

[0009]

[0010] The steps include:

[0011] (1) Synthesis of dibasic acid anhydride: reacting 2,2'-dithiodiacetic acid with acetic anhydride to obtain dibasic acid anhydride; after the reaction is completed, removing excess acetic anhydride under reduced pressure;

[0012] (2) Synthesis of fatty alcohol monoester: Under the action of a catalyst 4-dimethylaminopyridine, the dibasic acid anhydride obtained in step (1) is subjected to an esterification reaction with heptyl undecyl alcohol; after separation and purification, an intermediate product, heptyl undecyl alcohol monoester, is obtained;

[0013] (3) Synthesis of SN38-fatty alcohol prodrug: The heptyl undecyl alcohol monoester obtained in step (2) is dissolved in chloroform, and then a catalyst dissolved in chloroform is added dropwise, and activated in an ice bath. After the activation, SN38 dissolved in DMSO is added for esterification reaction, and SN38-fatty alcohol prodrug is obtained by separation and purification.

[0014] Preferably, in step (1), the molar ratio of the 2,2'-dithiodiacetic acid to acetic anhydride is 1:1-50.

[0015] Preferably, in step (2), the molar ratio of 4-dimethylaminopyridine: heptyl undecyl alcohol: dibasic acid anhydride is 1:(1-10):(1-15).

[0016] Preferably, in step (2), the esterification reaction is carried out under nitrogen protection conditions.

[0017] Preferably, in step (3), the catalyst is one or a combination of two or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 4-dimethylaminopyridine (DMAP), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and p-toluenesulfonic acid.

[0018] Preferably, in step (3), the activation is carried out under nitrogen protection for 0.5-2h; the esterification reaction is specifically carried out at room temperature for 36-48h under nitrogen protection; the separation and purification is specifically carried out by preparative liquid phase separation and purification to obtain the final product SN38-fatty alcohol prodrug, and the mobile phase system is water and acetonitrile (1‰ formic acid).

[0019] Preferably, in the step (3), the final product is obtained by separation and purification through preparative liquid phase, and the mass purity of the final product is above 99%.

[0020] The present invention also provides a method for preparing self-assembled nanoparticles of SN38-fatty alcohol prodrug, comprising the following steps:

[0021] The SN38-fatty alcohol prodrug is dissolved in an organic solvent, or the PEG modifier, the active targeting modifier and the SN38-fatty alcohol prodrug are dissolved in an organic solvent, and the solution is slowly added dropwise to water under stirring to spontaneously form uniform self-assembled nanoparticles, and the organic solvent is removed by a reduced pressure rotary evaporation method to obtain self-assembled nanoparticles of the SN38-fatty alcohol prodrug;

[0022] Among them, the mass ratio of the mixture of SN38-fatty alcohol prodrug: PEG modifier and active targeting modifier is 1:(0.1-1), preferably 1:(0.1-0.3), and more preferably 1:0.2; in the mixture of PEG modifier and active targeting modifier, the mass ratio of PEG modifier: active targeting modifier is (0-1):(1-0), preferably (1-3):(3-1), and more preferably 1:3.

[0023] Preferably, the organic solvent is acetone, DMF, DMSO, preferably DMF.

[0024] Preferably, the PEG modifier comprises an amphiphilic polymer, and the amphiphilic polymer is DSPE-PEG, TPGS, PEG-PLGA, PEG-PE, preferably DSPE-PEG.

[0025] Furthermore, the molecular weight of PEG in the DSPE-PEG is 1000-20000, preferably 2000.

[0026] Preferably, the active targeting modifier is an amphiphilic polymer modified with a target head molecule capable of targeting a specific tissue, wherein the target head molecule is Sialic acid, Biotin, Lipoic acid, cRGD, Folic acid, Galacto- ose, Glucose, Mannose, Hyaluronic acid; preferably, the active targeting modifier is DSPE-PEG-Folicacid, and the molecular weight of PEG is 1000-20000, more preferably, the molecular weight is 2000.

[0027] Preferably, the particle size of the self-assembled nanoparticles of the SN38-fatty alcohol prodrug is 80-200 nm, the particle size distribution is less than 0.3, and the drug loading is 19-50%.

[0028] The present invention also provides self-assembled nanoparticles of SN38-fatty alcohol prodrug prepared by the preparation method.

[0029] The present invention also provides the use of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in the preparation of anti-tumor drugs.

[0030] The present invention also provides the use of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in the preparation of an injection, oral administration or local administration system.

[0031] The use of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in preparing a drug delivery system.

[0032] Beneficial effects of the present invention:

[0033] (1) The present invention designs and synthesizes a SN38-fatty alcohol prodrug, and the synthesis method is simple and easy; and prepares a series of self-assembled nanoparticles of SN38-fatty alcohol prodrug with small particle size and uniform particle size distribution, and the preparation method is simple and easy;

[0034] (2) The pharmaceutical properties and cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with different proportions of active targeting modifiers were investigated. It was found that the stability of prodrug self-assembled nanoparticles modified with targeting modifiers alone was poor. As the proportion of active targeting modifiers continued to increase, the proportion of excipients was too high and the cytotoxicity of the nanoparticles was not significantly improved.

[0035] (3) The effects of co-modification of different ratios of PEG and active targeting modifiers on the pharmaceutical properties and anti-tumor activity of prodrug self-assembled nanoparticles were investigated. The results showed that the self-assembled nanoparticles of SN38-fatty alcohol prodrug co-modified with PEG and active targeting modifiers can effectively improve the efficacy of SN38 and reduce its toxic side effects; different ratios of surface functionalized module composition will have a significant effect on the pharmaceutical properties and cytotoxicity of the self-assembled nanoparticles of SN38-fatty alcohol prodrug. Compared with 20% PEG NPs and 20% FA NPs, 15% FA+5% PEG NPs has the best anti-tumor effect, successfully solving the paradox of the targeting effect and in vitro and in vivo stability of active targeting preparations; the present invention provides a new strategy and choice for the development of high-efficiency and low-toxic active targeting prodrug self-assembled nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the mass spectrum of the SN38-fatty alcohol prodrug in Example 1 of the present invention.

[0037] Figure 2 is the SN38-fatty alcohol prodrug in Example 1 of the present invention 1 H-NMR spectrum.

[0038] Figure 3 This is a purity diagram of the SN38-fatty alcohol prodrug in Example 1 of the present invention.

[0039] Figure 4 The graphs are the particle size changes of the prodrug self-assembled nanoformulations in Examples 2 and 4-6 of the present invention at room temperature, 4° C. refrigerated stability, and 37° C. shaking stability.

[0040] Figure 5 The cytotoxicity graphs of the prodrug self-assembled nanoformulation, SN38 solution, and irinotecan solution in Examples 4-6 of the present invention are shown.

[0041] Figure 6 The graphs are the particle size changes of the prodrug self-assembled nanoformulations in Examples 3, 8-10 of the present invention at room temperature, 4°C refrigerated stability, and 37°C shaking stability.

[0042] Figure 7 Transmission electron microscopy images of the prodrug self-assembled nanoformulations in Examples 2-4 and 8-10 of the present invention.

[0043] Figure 8 These are the cytotoxicity graphs of the prodrug self-assembled nanoformulations, SN38 solution, and irinotecan solution in Examples 3-4 and 8-10 of the present invention.

[0044] Fig. 9 This is a hemolysis test diagram of the prodrug self-assembled nanoformulation in Examples 3-4 and 10 of the present invention.

[0045] Fig.10 It is a blood drug concentration-time curve of the prodrug in the in vivo pharmacokinetic experiment of the prodrug self-assembled nanoparticles in Examples 3-4 and 10 of the present invention.

[0046] Fig.11 This is a blood drug concentration-time curve of the parent drug (SN38) in the in vivo pharmacokinetic experiment of the prodrug self-assembled nanoparticles in Examples 3-4 and 10 of the present invention.

[0047] Fig.12 This is a blood drug concentration-time curve of the sum of the prodrug and the parent drug in the in vivo pharmacokinetic experiment of the prodrug self-assembled nanoparticles in Examples 3-4 and 10 of the present invention.

[0048] Fig.13 The graphs show the changes in tumor volume in the in vivo anti-tumor experiment of the prodrug self-assembled nanoformulations, SN38 solution, and irinotecan solution in Examples 3-4 and 10 of the present invention administered at a low dose (3 mg / kg) (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0049] Fig.14 Figure 3 is a graph showing changes in mouse body weight in an in vivo anti-tumor experiment of the prodrug self-assembled nanoformulations, SN38 solution, and irinotecan solution of Examples 3-4 and 10 of the present invention administered at a low dose (3 mg / kg) (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0050] Fig.15 Comparison of tumor burden rates of the prodrug self-assembled nanoformulations in Examples 3-4 and 10 of the present invention, SN38 solution, and irinotecan solution in in vivo anti-tumor experiments administered at a low dose (3 mg / kg) (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0051] Fig.16The graphs show the changes in tumor volume of the prodrug self-assembled nanoformulations, SN38 solution, and irinotecan solution in Examples 3-4 and 10 of the present invention administered at a high dose (6 mg / kg) in the in vivo anti-tumor experiment (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0052] Fig.17 Figure 3 is a graph showing changes in mouse body weight in an in vivo anti-tumor experiment of the prodrug self-assembled nanoformulations, SN38 solution, and irinotecan solution of Examples 3-4 and 10 of the present invention administered at a high dose (6 mg / kg) (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0053] Fig.18 Comparison of tumor burden rates of the prodrug self-assembled nanoformulations in Examples 3-4 and 10 of the present invention, SN38 solution, and irinotecan solution in in vivo anti-tumor experiments administered at a high dose (6 mg / kg) (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). DETAILED DESCRIPTION

[0054] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0055] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0056] Embodiment 1:

[0057] Synthesis of SN38-fatty alcohol prodrugs with 2,2'-dithiodiacetic acid as a linker

[0058] Weigh 1g (5.5mmol) of 2,2'-dithiodiacetic acid and add 15mL of acetic anhydride. After it is completely dissolved, stir and react at room temperature for 2h. After the reaction is completed, add azeotropic toluene in small amounts and multiple times, and use a rotary evaporator to remove toluene and high-boiling acetic anhydride under reduced pressure to obtain a light yellow oily liquid dithiodiacetic anhydride. Add an appropriate amount of anhydrous dichloromethane to dissolve the dithiodiacetic anhydride formed, and slowly add 1.3g (3.66mmol) of heptyl undecyl alcohol solution dissolved in anhydrous dichloromethane and 85.4mg (0.7mmol) of 4-dimethylaminopyridine (DMAP) under stirring. React at room temperature for 12h, and detect the reaction progress by thin layer chromatography. After the reaction is completed, a crude product of heptyl undecyl alcohol-dithiodiacetic acid monoester is obtained. Separation and purification are performed by column chromatography, and the elution system is cyclohexane-ethyl acetate plus 0.1% glacial acetic acid.

[0059] Weigh 110.74 mg (0.255 mmol) of heptyl undecyl alcohol-dithiodiacetic acid monoester and dissolve it in chloroform, add dropwise 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 97.77 mg (0.51 mmol), 1-hydroxybenzotriazole 34.46 mg (0.255 mmol), and 4-dimethylaminopyridine 9.346 mg (0.0765 mmol) dissolved in chloroform, and activate in an ice bath for 2 h. Then add SN38 100 mg (0.255 mmol) dissolved in DMSO, and react at room temperature for 48 h. After the reaction, separate and purify the target product using the preparation phase to obtain SN38-fatty alcohol prodrug.

[0060] The product structure was confirmed by mass spectrometry. Figure 1 As shown, MS (ESI) m / z for C 44 H 60 N2O8S2[M+Na] + :808.38.

[0061] use 1 H-NMR confirmed the structure of the product. Figure 2 ). The results of hydrogen spectrum analysis are as follows:

[0062] 1H NMR (400MHz, DMSO-d6) δ8.24(d,J=9.1Hz,1H,Ar-H),8.03(d,J=2.5Hz,1H,Ar-H),7.68(dd,J=9.2,2.4Hz,1H,Ar -H),7.34(s,1H,Ar-H),6.50(s,1H,20-OH),5.44(s,2H,-CH2-),5.34(s,2H,-CH2-),4.10(s,2H,-CH2-),3.98( d,J=5.6Hz,2H,-CH2-),3.30(s,2H),3.20(d,J=7.7Hz,2H,-CH2-),1.88(p,J=6.9Hz,2H,-CH2-),1.31(t,J=7.6 Hz,3H,7-CH3),1.21(dd,J=23.2,4.3Hz,29H,-CH2-),0.89(d,J=7.2Hz,3H,8-CH3),0.81(t,J=6.8Hz,6H,-CH3).

[0063] The purity of SN38-fatty alcohol prodrug was 99.40% as analyzed by HPLC. Figure 3 shown.

[0064] Embodiment 2:

[0065] Preparation of non-PEGylated SN38-fatty alcohol prodrug self-assembled nanoparticles (0% PEG NPs)

[0066] 2 mg of the prodrug in Example 1 was accurately weighed and dissolved in 100 μL DMF. The DMF solution was slowly added dropwise to 2 mL of deionized water under stirring to spontaneously form non-PEGylated SN38-fatty alcohol prodrug self-assembled nanoparticles with uniform particle size, a particle size of 133.1±4.258 nm, a PDI of 0.200±0.008, and a potential of -18.6 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows: Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The placement stability of 0% PEG NPs was investigated in a shaking environment at room temperature, 4°C, and 37°C. Figure 4 As shown, the particle size increased significantly after being placed at room temperature for 12 days, at 4°C for 12 days, and in a shaking environment at 37°C for 12 days.

[0067] Embodiment 3:

[0068] Preparation of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles (20% PEG NPs)

[0069] Accurately weigh 2 mg of the prodrug in Example 1, 0.5 mg of DSPE-PEG 2000 , dissolved in 100 μL DMF, and slowly added the DMF solution to 2 mL deionized water under stirring, spontaneously forming nanoparticles with uniform particle size, with a particle size of 90.43±1.155 nm, a PDI of 0.243±0.024, and a potential of -26.2 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows: Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The placement stability of 20% PEG NPs was investigated at room temperature, 4°C and 37°C in a shaker environment. Figure 6 As shown, there was no significant change in particle size after being placed at room temperature for 11 days, at 4°C for 11 days, and in a shaking environment at 37°C for 11 days.

[0070] Embodiment 4:

[0071] Preparation of Active Targeting Modified SN38-Fatty Alcohol Prodrug Self-Assembled Nanoparticles (20% FA NPs)

[0072] Accurately weigh 2 mg of the prodrug in Example 1, 0.5 mg of DSPE-PEG 2000 -Folic acid was dissolved in 100 μL DMF, and the DMF solution was slowly added dropwise to 2 mL deionized water under stirring, spontaneously forming nanoparticles with uniform particle size, with a particle size of 119.6±2.495 nm, a PDI of 0.151±0.033, and a potential of -8.06 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The placement stability of 20% FA NPs was investigated at room temperature, 4°C and 37°C in a shaker environment. Figure 4 As shown, the particle size increased significantly after being placed at room temperature for 12 days, at 4°C for 12 days, and in a shaking environment at 37°C for 12 days.

[0073] Embodiment 5:

[0074] Preparation of Active Targeting Modified SN38-Fatty Alcohol Prodrug Self-Assembled Nanoparticles (40% FA NPs)

[0075] Accurately weigh 2 mg of the prodrug in Example 1, 1.33 mg of DSPE-PEG 2000-Folic acid was dissolved in 100 μL DMF, and the DMF solution was slowly added to 2 mL of deionized water under stirring, spontaneously forming nanoparticles with uniform particle size of 131.2±4.067 nm, PDI of 0.151±0.033, and potential of -1.62 mV. The placement stability of 40% FANPs was investigated at room temperature, 4°C, and 37°C in a shaker environment. Figure 4 As shown, the particle size increased significantly after being placed at room temperature for 12 days, at 4°C for 12 days, and in a shaking environment at 37°C for 12 days.

[0076] Embodiment 6:

[0077] Preparation of SN38-fatty alcohol prodrug self-assembled nanoparticles (60% FA NPs) using targeting modifier alone

[0078] Accurately weigh 2 mg of the prodrug in Example 1 and 3 mg of DSPE-PEG 2000 -Folic acid was dissolved in 100 μL DMF, and the DMF solution was slowly added to 2 mL of deionized water under stirring, spontaneously forming nanoparticles with uniform particle size of 136.6±1.922 nm, PDI of 0.206±0.011, and potential of 0.330 mV. The placement stability of 60% FA NPs was investigated at room temperature, 4°C, and 37°C in a shaker environment. Figure 4 As shown, the particle size increased significantly after being placed at room temperature for 12 days, at 4°C for 12 days, and in a shaking environment at 37°C for 12 days.

[0079] Table 1. Particle size, size distribution, surface charge and drug loading of non-PEG-modified and actively targeted SN38-fatty alcohol prodrug self-assembled nanoparticles

[0080]

[0081] The results of Example 2 and Examples 4-6 show that when the mass ratio of the active targeting modifier in the prodrug self-assembled nanoformulation is regulated, the prodrug molecule has a strong assembly ability, and the particle size is between 90-140nm, which is conducive to tumor-targeted accumulation through the high permeability and long retention effect of solid tumors. At the same time, with the increase in the proportion of folic acid modifiers, the potential of the prodrug self-assembled nanoformulation gradually increases. 60% FA NPs even present a positively charged Zeta potential, which is not conducive to preventing the aggregation of nanoparticles through charge repulsion. In the shaking environment of room temperature, 4°C, and 37°C, each group of preparations has different degrees of particle size increase and poor stability.

[0082] Embodiment 7:

[0083] Cytotoxicity of SN38-aliphatic alcohol prodrug self-assembled nanoparticles with active targeting modifiers

[0084] The MTT method was used to investigate the cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with active targeting modifiers on mouse breast cancer cells (4T1 cells) and mouse colon cancer cells (CT26 cells). First, the cells in good condition were digested and diluted to 2×10 4 cells / mL, and after the cells are evenly blown, 100 μL of cell suspension is added to each well of a 96-well plate, and the plates are placed in an incubator at 37°C and 5% CO2 for 24 hours to allow them to adhere to the wall. After the cells adhere to the wall, SN38 solution or irinotecan solution or the prodrug self-assembled nanoparticles prepared in Examples 4-6 are added. In this experiment, the preparation and dilution of the drug solution and the prodrug self-assembled nanoparticle preparations were all made with the corresponding culture fluid, and aseptically filtered with a 0.22 μm filter membrane. 100 μL of the test solution was added to each well, with 3 parallel wells for each concentration. For the control group, no test solution was added, and 100 μL of culture fluid was added alone, and the cells were incubated together in an incubator. 48 hours after drug addition, the 96-well plate was taken out, 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was shaken after incubation in an incubator for 4 hours. After the 96-well plate was inverted on filter paper to fully absorb the residual liquid, 200 μL DMSO was added to each well and oscillated on an oscillator for 15 minutes to fully dissolve the blue-purple crystals. Set A1 well (containing only 200 μL DMSO) as the zeroing well. Use an ELISA reader to measure the absorbance value of each well after zeroing at 570 nm.

[0085] The results of the cytotoxicity experiments were analyzed and the half inhibitory concentration (IC 50 ). Compared with the SN38 solution, the cytotoxicity of irinotecan solution and prodrug nanoparticles is reduced, because it takes a certain amount of time for SN38 to be released from the prodrug nanoparticles. The cytotoxicity of irinotecan solution is the lowest, indicating that its conversion into active SN38 is less efficient. In contrast, the cytotoxicity of prodrug nanoparticles is stronger, but with the increase of the folic acid modification ratio, the cytotoxicity of prodrug nanoparticles modified with active targeting modifiers does not increase significantly.

[0086] Table 2. Cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with active targeting modifiers

[0087]

[0088] Embodiment 8:

[0089] Preparation of SN38-fatty alcohol prodrug self-assembled nanoparticles (5% FA + 15% PEG NPs) co-modified with PEG and active targeting modifiers

[0090] Accurately weigh 2 mg of the prodrug in Example 1 and 0.125 mg of DSPE-PEG 2000 -Folic acid, 0.375mg DSPE-PEG 2000 , dissolved in 100 μL DMF, and slowly added the DMF solution to 2 mL deionized water under stirring, spontaneously forming nanoparticles with uniform particle size, with a particle size of 89.9±0.9885 nm, a PDI of 0.120±0.070, and a potential of -22.7 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows: Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The storage stability of 5% FA + 15% PEG NPs was investigated in a shaking environment at room temperature, 4°C and 37°C. Figure 6 As shown, there was no significant change in particle size after being placed at room temperature for 11 days, at 4°C for 11 days, and in a shaking environment at 37°C for 11 days.

[0091] Embodiment 9:

[0092] Preparation of SN38-fatty alcohol prodrug self-assembled nanoparticles (10% FA + 10% PEG NPs) co-modified with PEG and active targeting modifiers

[0093] Accurately weigh 2 mg of the prodrug in Example 1, 0.25 mg of DSPE-PEG 2000 -Folic acid, 0.25 mg DSPE-PEG 2000 , dissolved in 100 μL DMF, and slowly added the DMF solution to 2 mL deionized water under stirring, spontaneously forming nanoparticles with uniform particle size, with a particle size of 90.86±1.001 nm, a PDI of 0.161±0.043, and a potential of -17.1 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows: Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The storage stability of 10% FA + 10% PEG NPs was investigated in a shaking environment at room temperature, 4°C and 37°C. Figure 6 As shown, there was no significant change in particle size after being placed at room temperature for 11 days, at 4°C for 11 days, and in a shaking environment at 37°C for 11 days.

[0094] Embodiment 10:

[0095] Preparation of SN38-fatty alcohol prodrug self-assembled nanoparticles (15% FA + 5% PEG NPs) co-modified with PEG and active targeting modifiers

[0096] Accurately weigh 2 mg of the prodrug in Example 1 and 0.375 mg of DSPE-PEG 2000 -Folic acid, 0.125mg DSPE-PEG 2000 , dissolved in 100 μL DMF, and slowly added the DMF solution to 2 mL deionized water under stirring, spontaneously forming nanoparticles with uniform particle size, with a particle size of 99.96±3.193 nm, a PDI of 0.230±0.014, and a potential of -15.7 mV. The particle size and morphology of the prepared prodrug nanoparticles were measured by transmission electron microscopy, and the results were as follows: Figure 7 The transmission electron microscopy images show that the prepared prodrug self-assembled nanoparticles are uniformly spherical. The storage stability of 15% FA + 5% PEG NPs was investigated in a shaking environment at room temperature, 4°C and 37°C. Figure 6 As shown, there was no significant change in particle size after being placed at room temperature for 11 days, at 4°C for 11 days, and in a shaking environment at 37°C for 11 days.

[0097] Table 3. Particle size, particle size distribution, surface charge and drug loading of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with PEG or co-modified with PEG and active targeting modifiers

[0098]

[0099] The results of Examples 8-10 show that when PEG is co-modified with an active targeting modifier and the DSPE-PEG in the modifier is regulated 2000 DSPE-PEG 2000 -Folic acid, the prodrug molecules have strong self-assembly properties, and the particle size of the prepared prodrug nanoparticles is less than 120nm, which is conducive to tumor targeted accumulation through the high permeability and long retention effect of solid tumors. At the same time, the potential of the SN38-fatty alcohol prodrug self-assembled nanoparticles co-modified with PEG and active targeting modifiers is between -15mV and 30mV, which is conducive to preventing the aggregation of nanoparticles through charge repulsion. It has good room temperature placement, 4℃ stability, and 37℃ shaking stability. Among them, the particle size of 10% FA+10% PEG NPs and 15% FA+5% PEG NPs preparations is the most stable.

[0100] Embodiment 11:

[0101] Cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with PEG or co-modified with PEG and active targeting modifiers

[0102] The MTT method was used to investigate the cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with PEG or co-modified with PEG and active targeting modifiers on mouse breast cancer cells (4T1 cells), mouse colon cancer cells (CT26 cells), and human non-small cell lung cancer (A549 cells). First, the cells in good condition were digested and diluted to 2×10 4 cells / mL, after the cells are evenly blown, 100 μL of cell suspension is added to each well of a 96-well plate, and the plates are placed in an incubator at 37°C and 5% CO2 for 24 hours to allow them to adhere to the wall. After the cells adhere to the wall, SN38 solution or irinotecan solution or the prodrug self-assembled nanoparticles prepared in Examples 3-4 and 8-10 are added. In this experiment, the preparation and dilution of the drug solution and the prodrug self-assembled nanoparticle preparations are all made with the corresponding culture fluid, and are aseptically filtered with a 0.22 μm filter membrane. 100 μL of the test solution is added to each well, with 3 parallel wells for each concentration. For the control group, no test solution is added, and a single 100 μL culture fluid is added, and the cells are incubated together in an incubator. 48 hours after drug addition, the 96-well plate was taken out, 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was shaken after incubation in an incubator for 4 hours. After the 96-well plate was inverted on filter paper to fully absorb the residual liquid, 200 μL DMSO was added to each well and oscillated on an oscillator for 15 minutes to fully dissolve the blue-purple crystals. Set A1 well (containing only 200 μL DMSO) as the zeroing well. Use an ELISA reader to measure the absorbance value of each well after zeroing at 570 nm.

[0103] The results of the cytotoxicity experiments were analyzed and the half inhibitory concentration (IC 50 ). Compared with the SN38 solution, the cytotoxicity of irinotecan solution and prodrug nanoparticles was reduced, because it takes a certain amount of time for SN38 to be released from the prodrug nanoparticles. The cytotoxicity of irinotecan solution was the lowest, indicating that its conversion into active SN38 was less efficient. In contrast, the prodrug nanoparticles were more cytotoxic. Among the five prodrug nanoparticles, the cytotoxicity of the prodrug nanoformulations gradually increased with the increase of the folic acid modification ratio. 20% FA NPs had higher cytotoxicity to tumor cells, followed by 15% FA + 5% PEG NPs.

[0104] Table 4. Cytotoxicity of SN38-fatty alcohol prodrug self-assembled nanoparticles modified with PEG or co-modified with PEG and active targeting modifiers

[0105]

[0106] Example 12: In vitro hemolysis experiment of SN38-fatty alcohol prodrug self-assembled nanoparticles

[0107] Fresh red blood cell suspension was mixed with SN38 solution or irinotecan solution or the prodrug self-assembled nanoformulation prepared in Examples 3-4 and 10 at a ratio of 1:19, and a positive control group (20% TritonX-100) and a negative control group (PBS solution) were set up respectively, and incubated in a shaker at 37°C for 1 hour. The red blood cells were then centrifuged at low speed (3000 rpm, 10 min). The results are as follows: Fig. 9 As shown, after the SN38 solution was incubated with the red blood cell suspension, the red blood cells ruptured and the supernatant was obviously blood red, while after the prodrug self-assembled nanoformulations of Examples 3-4 and 10 were incubated with the red blood cell suspension, the supernatant was clear and blood-free by naked eye observation. Further, the supernatant was added to a 96-well plate and the absorbance was measured at 540 nm to calculate the hemolysis rate ((absorbance A 制剂 -A 阴性 ) / (A 阳性 -A 阴性 )*100%), the results are shown in Table 5.

[0108] Table 5. Hemolysis rate test results of SN38 solution, irinotecan solution, Examples 3-4, and 10

[0109]

[0110] The results show that the SN38 solution can cause hemolysis, with a hemolysis fraction of 3.955%, while the prodrug self-assembled nanoformulations of Examples 3, 4, and 10 can significantly improve the hemolysis, with a hemolysis fraction of less than 0.8%, and have good biocompatibility and safety.

[0111] Embodiment 13:

[0112] Pharmacokinetics of SN38-fatty alcohol prodrug self-assembled nanoparticles

[0113] Thirty male SD rats (180-220 g) were randomly divided into five groups. The rats were fasted for 12 hours before administration and were allowed to drink water freely. The five groups of rats were intravenously injected with SN38 solution and irinotecan injection. The prodrug self-assembled nanoformulations prepared in Examples 3-4 and 10 were dosed at 5 mg / kg (equivalent to SN38 dose). Blood was collected from the eye sockets at the specified time points and centrifuged to obtain plasma. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

[0114] The experimental results are as follows Figure 10-12 , as shown in Table 6, Figure 10-12Represent the blood concentration curves of SN38 (or irinotecan, or SN38 released from the prodrug self-assembled nanoformulation), prodrug molecules (irinotecan / SN38-fatty alcohol prodrug) in the blood, and the sum of prodrug and SN38 (the sum of irinotecan / SN38-fatty alcohol prodrug and released SN38). SN38 solution is rapidly cleared in the body, with a short systemic circulation time and an average residence time of only 0.661 hours. As a hydrophilic prodrug of SN38, irinotecan has a reduced affinity for plasma protein binding, resulting in a slower blood clearance rate. Therefore, the area under the curve (AUC) of total SN38 in irinotecan is approximately 8.59 times that of SN38 solution. However, the presence of a large amount of carboxylesterase in the blood causes 27% of SN38 to be released from irinotecan solution, which may lead to severe blood toxicity.

[0115] Compared with the two solutions, the three prodrug self-assembled nanoformulations significantly prolonged the blood circulation time of SN38. Among them, due to the good colloidal stability of 15% FA+5% PEG NPs and 20% PEG NPs, the AUC of the total SN38 of the two increased by about 212 times and 191 times compared with the SN38 solution. And only no more than 5% of the free SN38 was released by the two, which effectively prevented the exposure of the drug in the blood circulation, thereby significantly reducing the hematological toxicity associated with SN38. Compared with the two, the colloidal stability of 20% FA NPs is poor and it is easy to disintegrate in the blood circulation. Without the protection of "invisible molecules", the 20% FA NPs covered with folic acid are easily recognized and adsorbed by immunoglobulins in the blood, thereby accelerating the blood clearance of the preparation and the phagocytosis of macrophages. Therefore, although the prodrug self-assembly strategy of 20% FA NPs prolonged the systemic circulation time of SN38 to some extent, its total SN38 AUC was significantly reduced compared with that of 15% FA + 5% PEG NPs and 20% PEG NPs, which was only increased by about 118 times compared with the SN38 solution.

[0116] Table 6. Pharmacokinetic parameters of prodrug self-assembled nanoformulations

[0117]

[0118] Embodiment 14:

[0119] In vivo antitumor experiment of SN38-fatty alcohol prodrug self-assembled nanoparticles

[0120] Mouse breast cancer cell suspension (4T1, 3×10 6 cells / 100 μL) were inoculated subcutaneously on the dorsal flank of female Balb / c mice. 3At about 14 days, the tumor-bearing mice were randomly divided into groups, with 5 mice in each group. SN38 solution and irinotecan injection were administered intravenously every three days. The prodrug self-assembled nanoformulations prepared in Examples 3-4 and 10 were used as negative control groups. As the positive control group, the drug was administered 4 times, with a dose of 3 mg / kg (low dose, calculated as SN38), and the tumor volume and mouse body weight were measured every day. At the same time, the dose was increased to 6 mg / kg (high dose, calculated as SN38) to evaluate the SN38 solution and irinotecan injection And the tolerability and anti-tumor activity of the prodrug self-assembled nanoformulations prepared in Examples 3-4 and 10. The administration cycle and the number of administrations were consistent with those of the low-dose group.

[0121] The results of low-dose and high-dose treatments are as follows Figure 13-18 As shown. The saline group, which served as a negative control, had rapid tumor progression and the highest tumor burden rate in mice. Compared with the saline group, both the SN38 solution and the irinotecan solution showed a certain degree of tumor growth inhibition. However, their therapeutic effects were limited by their rapid clearance and poor tumor accumulation, and the low conversion efficiency of irinotecan further limited its anti-tumor effect. In contrast, the three groups of prodrug self-assembled nanoformulations showed better tumor inhibition than the SN38 solution, although they showed lower cytotoxicity in vitro. This enhanced anti-tumor effect can be attributed to the extended blood circulation time and enhanced tumor accumulation of the nanoformulations. Among them, due to the poor pharmacokinetic behavior of 20% FA NPs, its tumor inhibition effect was significantly weaker than that of 15% FA+5% PEGNPs and 20% PEG NPs. Due to the targeting effect of folic acid molecules on tumors, even though there was no significant difference in the pharmacokinetic behavior between 15% FA + 5% PEG NPs and 20% PEG NPs, 15% FA + 5% PEG NPs showed a stronger anti-tumor effect.

[0122] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A SN38-fatty alcohol prodrug, characterized in that: Its structure is shown in formula (I):

2. The method for synthesizing the SN38-fatty alcohol prodrug according to claim 1, characterized in that: The synthetic route is as follows: The steps include: (1) Synthesis of dibasic acid anhydride: reacting 2,2'-dithiodiacetic acid with acetic anhydride to obtain dibasic acid anhydride; after the reaction is completed, removing excess acetic anhydride under reduced pressure; (2) Synthesis of fatty alcohol monoester: Under the action of a catalyst 4-dimethylaminopyridine, the dibasic acid anhydride obtained in step (1) is subjected to an esterification reaction with heptyl undecyl alcohol; after separation and purification, an intermediate product, heptyl undecyl alcohol monoester, is obtained; (3) Synthesis of SN38-fatty alcohol prodrug: The heptyl undecyl alcohol monoester obtained in step (2) is dissolved in an organic solvent, and then a catalyst dissolved in an organic solvent is added dropwise, and activated in an ice bath. After the activation, SN38 dissolved in DMSO is added for esterification reaction, and SN38-fatty alcohol prodrug is obtained by separation and purification.

3. The synthesis method according to claim 2, characterized in that In step (1), the molar ratio of the 2,2'-dithiodiacetic acid to acetic anhydride is 1:1-50; in step (2), the molar ratio of the 4-dimethylaminopyridine:heptylundecanol:dibasic acid anhydride is 1:(1-10):(1-15); in step (3), the catalyst is one or a combination of two or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 4-dimethylaminopyridine (DMAP), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and p-toluenesulfonic acid; the organic solvent is chloroform, the activation is carried out under nitrogen protection, and the activation is carried out for 0.5-2h; the esterification reaction is specifically carried out at room temperature under nitrogen protection for 36-48h.

4. A method for preparing self-assembled nanoparticles of SN38-fatty alcohol prodrug, characterized in that: The steps include: Dissolving the SN38-fatty alcohol prodrug of claim 1 in an organic solvent, or dissolving a PEG modifier, an active targeting modifier, and the SN38-fatty alcohol prodrug of claim 1 in an organic solvent, slowly dropping the solution into water under stirring to spontaneously form uniform self-assembled nanoparticles, and removing the organic solvent to obtain self-assembled nanoparticles of the SN38-fatty alcohol prodrug; Among them, the mass ratio of the mixture of SN38-fatty alcohol prodrug: PEG modifier and active targeting modifier is 1:(0.1-1), preferably 1:(0.1-0.3), and more preferably 1:0.2; in the mixture of PEG modifier and active targeting modifier, the mass ratio of PEG modifier: active targeting modifier is (0-1):(1-0), preferably (1-3):(3-1), and more preferably 1:

3.

5. The preparation method according to claim 4, characterized in that: The organic solvent is acetone, DMF, DMSO, preferably DMF; the PEG modifier includes an amphiphilic polymer, and the amphiphilic polymer is DSPE-PEG, TPGS, PEG-PLGA, PEG-PE, preferably DSPE-PEG; the active targeting modifier is an amphiphilic polymer modified with a target head molecule, wherein the target head molecule is Sialic acid, Biotin, Lipoic acid, cRGD, Folic acid, Galactose, Glucose, Mannose, and Hyaluronic acid.

6. The preparation method according to claim 5, characterized in that: The molecular weight of PEG in the DSPE-PEG is 1000-20000, preferably 2000; the active targeting modifier is DSPE-PEG-Folic acid, and the molecular weight of PEG is 1000-20000, preferably 2000.

7. Self-assembled nanoparticles of SN38-fatty alcohol prodrug prepared by the preparation method according to any one of claims 4 to 6.

8. Use of the self-assembled nanoparticles of the SN38-fatty alcohol prodrug according to claim 1 or the SN38-fatty alcohol prodrug according to claim 7 in the preparation of anti-tumor drugs.

9. Use of the self-assembled nanoparticles of the SN38-fatty alcohol prodrug according to claim 1 or the SN38-fatty alcohol prodrug according to claim 7 in the preparation of an injection, oral administration or topical administration system.

10. Use of the self-assembled nanoparticles of the SN38-fatty alcohol prodrug according to claim 1 or the SN38-fatty alcohol prodrug according to claim 7 in preparing a drug delivery system.

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